Stem cells are immature, unspecialized cells that renew themselves for long periods through cell division. Under certain conditions, they can differentiate into mature, functional cells. Human embryonic stem cells (hESC) are derived from early surplus human blastocysts 1,2. Human ES cells are unique stem cells since they can self-renew infinitely in culture, and since they have a remarkable potential to develop into extraembryonic lineages as well as all somatic cells and tissues of the human body1,2.
Given the unique properties of hESC, they are expected to have far-reaching applications in the areas of basic scientific research, pharmacology, and regenerative medicine. Human ES cell lines can provide a powerful in vitro model for the study of the molecular and cellular biology of early human development, for functional genomics, drug screening, and discovery. They may serve for toxicology and teratogenicity high throughput screening. Since hESC can self-renew indefinitely and can differentiate into any cell type, they can serve as a renewable, unlimited donor source of functionally mature differentiated cells or tissues for transplantation therapy. In addition, transplanted genetically-modified hESC can serve as vectors to carry and express genes in target organs in the course of gene therapy.
While the promise of hESC for basic scientific research pharmacology and regenerative medicine is remarkable, the exploitation of hESC for most applications depends upon further development. Improved control of the growth of undifferentiated hESC, the development of bulk feeder-free cultures of undifferentiated cells, the development of animal-free culture systems, and the development of methods and tools which direct the differentiation and generate pure cultures of mature functional cells of a specific type are required.
At present, few culture systems are most commonly used to propagate undifferentiated hESC1-4. In the initial culture system that was developed, undifferentiated hESC are cultured in serum-containing medium as colonies, upon a layer of fibroblast feeder cells (of mouse1,2 or human origin5,11). It is possible to remove all animal products from this culture system and replace them with those from a human source6. It was found that in this system the cells are propagated as clumps on a low scale which does not allow cloning.
An alternative culture system that was developed and used extensively is a serum-free system that includes the knockout (KO) medium supplemented with knockout serum replacement (KOSR) and FGF2. This system allows cloning of undifferentiated hESC, although at a low efficiency3. Undifferentiated cells are cultured as flat colonies and may be propagated as small clusters or single cells (by using trypsin7).
Another alternative culture system for use in the proliferation of undifferentiated growth of hESC comprises a culture matrix comprising extracellular matrix (ECM) prepared from feeder cells and a conditioned medium being preconditioned by feeder cells. The suggested leading cells in the feeder cells include primary mouse embryonic fibroblasts (PMEF) a mouse embryonic fibroblast cell line (MEF) murine foetal fibroblasts (MFF) human embryonic fibroblasts (HEF) human foetal muscle (HFM) human foetal skin cells (HFS) human adult skin cells, human foreskin fibroblasts (HFF)10 human adult Fallopian tubal epithelial cells (HAFT) or human marrow stromal cells (HMSC).
Undifferentiated propagation may be accomplished with the KO serum-free culture system without the use of feeders by plating and growing colonies on extracellular matrices (ECM) within a feeder-conditioned KO medium supplemented with KOSR and FGF24. Furthermore, it has been suggested that feeder conditioning may be replaced by substituting the medium with high concentrations of FGF2 and noggin12. Alternatively, feeder conditioning was replaced by transforming growth factor β1 and human LIF (in addition to FGF2) and growing the cells on human fibronectin8. In a recent publication, undifferentiated propagation of hESC colonies, in the absence of feeders' was reported with a chemically defined medium without serum replacer, supplemented with activin or nodal plus FGF213.
A key limitation of hESC culture systems is that they do not allow the propagation of pure populations of undifferentiated stem cells and their use always involves some level of background differentiation. The stem cells most commonly follow a default pathway of differentiation into an epithelial cell type that grows either as a monolayer of flat squamous cells or form cystic structures. Most probably, this form of differentiation represents differentiation of hESC into extraembryonic endoderm9.
Spontaneous differentiation of hESC into presumably extraembryonic lineages also interferes with the derivation of somatic differentiated cells. Under various differentiation-inducing conditions, such as in embryoid bodies (EB) suspension cultures, differentiation into cystic extraembryonic structures may be common or may predominate and limit differentiation into somatic lineages. Control and elimination of the differentiation into extraembryonic lineages therefore, may be invaluable in the derivation of somatic lineages, in addition to its importance in maintaining the stem cells in an undifferentiated state. It has been recently demonstrated that under differentiation-inducing culture conditions, the bone morphogenetic protein (BMP) antagonist noggin can prevent extraembryonic differentiation of hESC and promote their differentiation into the neural lineage9.